The L-phenylalanine is an essential amino acid for humans and a raw material for production of aspartame, an artificial sweeter. The main industrial processes for phenylalanine production are enzymatic conversions and microbial fermentations. Because it is difficult to get the raw material for the enzymatic conversion, and the cost of the raw material changes significantly from time to time. Thus, the industries prefer to utilize microbial fermentations for phenylalanine production. Fed-batch operation is the most popular fermentation process, since it gives higher productivity than batch fermentationidoes, and it can also get rid of the high contamination problem of the continuous culture. However, as phenylalanine fermentation (see the work of Wang P.-M. et al, 1994, Biotechnology Techniques, V. 8 No. 11, November) was carried out by Corynebacterium qlutamicum, the product feedback inhibition by phenylalanine is still present. Besides, no work has mentioned the influence of oxygen supply rate on the product feedback inhibition of phenylalanine formation. The present inventions designs experiments to study the effect of oxygen supply rate on product feedback inhibition, and provides process control methods to decreases product feedback inhibition by proper increases of oxygen supply.
Some previous work in literature used phenylalanine resistant analogues such as phenylalanine p-fluorophenylalanine (p-FP), m-resistant fluorophenylalanine (m-FP), or aromtic amine analogues such as 3-amino-L-tyrosin (3AT), 5-methyltyryptophan (5MT) to screen high phenylalanine producing strains. The screened strains possess higher phenylalanine resistant capability, but phenylalanine production by the screened strains were still inhibited by high phenylalanine concentrations.
Some research work related to the recovery of phenylalanine from fermentation broth in past, such as using copper ions to bind phenylalanine to precipitate out of the solution, or using resin to absorb phenylalanine. But the conditions favoring recovery process do not favor fermentation process. Thus, the current technology still can not provide an economical process to recover phenylalanine from culture.
Thus, product feedback inhibition by phenylalanine is the problem of phenylalanine fermentation, which was alleviated by the present invention increasing oxygen supply rate during the course of fermentation.
Among the methods of controlling substrate feeding policy, the oxystat is an economical, automatic addition method. The oxystat is defined as feeding a limiting substrate only when dissolved oxygen tension increases due to substrate depletion (see the work of Yano T. et al., 1978, J. Ferment. Technol. 56, 416-420). Previous research pointed out that this method can effectively determine the timing of feeding substrate to maintain substrate concentration under low levels, which avoids inhibition of cell growth by high substrate concentrations. This method can also maintain culture under low dissolved oxygen tension. The oxystat has been used in phenylalanine production by recombinant Escherichia coli as well. However the final phenylalanine concentration was low due to the production of excess acetic acid under low oxygen tension which inhibited growth (Konstantinov K. et al., 1990, Biotechnol. Bioeng. 36, 750-758). Up to now, no successful attempts using an oxystat for a fed-batch phenylalanine fermentazion have been reported. This is also the problem in prior art of phenylalanine fermentation. The present invention solves this problem by successfully aplying an oxystat to phenylalanine fermentation by coryneform bacterium without production of inhibitory metabolites under low dissolved oxygen tension.
The information about phenylalanine production in literature is limited. As to the correlation between oxygen supply rate and phenylalanine production, it is an accepted concept that phenylalanine formation is favorable under low dissolved oxygen tension. Based on this concept, previous work reported in literature adopted the strategy of controlling culture under a lower oxygen supply rate or under a constant oxygen supply rate in the latter stages of phenylalanine fermentation in order to increase phenylalanine production. However, such strategy neglects the product feedback inhibition by high phenylalnine concentration. On the other hand, as opposite to the prior concept about the strategy of controlling the oxygen supply rate, the present invention provide methods to increase the oxygen supply rate at proper timing to increase fermentation productivity. Since product feedback inhibition is decreased by such process control method.
Thus, in order to improve phenylalanine fermentation productivity, the present invention provides methods to decrease product feedback inhibition by phenylalanine, and to keep culture under low dissolved oxygen tension without producing inhibitory metabolites and to keep substrate under low levels to avoid substrate inhibition on cell growth.